Transfusion of platelets (a commonly transfused cellular component of blood) is a cornerstone of modern medical care for a number of acute and chronic conditions characterized by either excessive bleeding or insufficiency of endogenous platelet production or function. Unlike red blood cells, which can be efficiently stored at 1-6° C. (mean 4° C.), platelets are irreversibly injured when temperatures repeatedly drop below approximately 20° C. for short periods of time or are kept at less than 20° C. for long periods of time. This injury is termed the “platelet cold storage lesion”. Importantly, this platelet cold storage lesion begins to occur even after brief exposure to temperatures less than 20° C. and is even seen in patients undergoing surgery in which the temperature of the whole body or of parts of the body is decreased to temperatures less than 20° C. and leads to bleeding abnormalities.
FIG. 1 depicts effects on platelets of cooling platelets from 37° C. to 4° C., in accordance with the related art. Exposure of platelets to temperatures less than 20° C. results in structural injury and functional activation of control (normal) platelets. In portion A of FIG. 1, significant morphological changes occur when platelets are cooled from 37° C. to 4° C. as shown by the appearance of filopodia using phase contrast microscopy. In portion B of FIG. 1, temperature dependent activation of platelets is further demonstrated by anti-phosphotyrosine Western Blot analysis of platelets incubated for 30 min at 37° C. (lanes 1, 3) or 4° C. (lanes 2, 4), in the absence (in lanes 1, 2) or presence (in lanes 3, 4) of a membrane-active compound. The blot was stripped and probed for actin as a loading control.
As shown in FIG. 1, key characteristics of this platelet cold storage lesion are: (1) reversible to irreversible morphological change from a discoid cell to spiculated spheres with protruding filopodia, depending on time at temperatures less than 20° C.; (2) irreversible immune-independent microaggregation of platelets (i.e., increased cell:cell interaction); (3) membrane clustering of the glycoprotein GPIb on the surface of platelets resulting in the formation of a neoantigen; and (4) subsequent recognition and phagocytosis by macrophages of the microaggregates and/or neoantigen-expressing platelets upon transfusion into a recipient. In addition, there is a significant reduction in circulation half-life of chilled platelets introduced into a recipient of the chilled platelets. As a consequence of this platelet cold storage lesion, platelets must be stored at 20-24° C. (mean of 22° C.) in order to maintain acceptable function and viability in the transfused patient (see American Association of Blood Banks (AABB) Technical Manual). Unfortunately, maintaining platelets at a mean temperature of 22° C. for prolonged periods of time greatly increases the risk of adverse medical events due to bacterial growth in the platelet product. Current estimates are that 1 in every 3000 platelet units are affected by microbial contamination (see Kleinman S H et al., “Two-year experience with aerobic culturing of apheresis and whole blood-derived platelets”, Transfusion 2006, 46:1787-1794). Risks are associated with transfusion of cellular blood components in Canada (see Transfusion Medicine Reviews, 17:120-163). Because of this microbial risk, platelets can only be stored at 20-24° C. for a maximum of 5 days before they must be destroyed.
Rosiello (International Publication No. WO 2006/044790 A2) discloses a method for the cold storage (−80° C. to 15° C.) of platelets for periods of 3 days to 28 days, by modifying the platelet membrane with a glycan-modifying agent, namely a sugar, a monosaccharide sugar, a nucleotide sugar, sialic acid, sialic acid precursors, CMP-sialic acid, UDP-galactose, and UDP-galactose precursors. Rosiello's method is not practical, however, because it is known that glycosylation (i.e., binding saccharides to proteins and/or lipids) fails to restore the functionality of chilled platelets in vivo.
For example, the inventors of the present invention were present at a seminar at the Center for Blood Research at the University of British Columbia on Apr. 26, 2006 at which Dr. Karin Hoffmeister gave a public presentation entitled “Platelet Glycosylation and the “In and Outs” of Platelet Transfusion” during which Dr. Hoffmeister talked about the problems that had been encountered with glycosylation, said problems including the fact that glycosylation does not protect platelets in chilled platelet concentrates. The results presented at the seminar were also published in a peer-review journal (Blood, 2008, 111: 3249-56).
In addition, Hans Wandall of Zymequest, Inc. gave a public presentation in California at the annual meeting of the California Blood Bank Society on Apr. 28, 2006 in which Hans Wandall substantiated that “glycosylation of platelets does not work, at least after extended storage in the cold and not for larger volumes,” which was confirmed by an attendee of said public presentation by Hans Wandall to an inventor of the present invention via email correspondence on Jun. 22, 2006.
“At a meeting of the American Society of Hematology on Dec. 11, 2006, S. J. Schlichter et al. reported the result of studies relating to galactosylated platelets derived from humans and stored a 4° C. and concluded: “The data show that, following two days of 4° C. storage, the recoveries and survivals of the galactosylated platelets are no different than the non-galactosylated 4° C. stored platelets from the same volunteer. Although the recoveries of the 4° C. stored platelets with and without galactosylation are well-maintained compared to the 22° C. stored platelets, the survivals are markedly reduced as had been previously shown for 4° C. stored platelets (Br J Haematol 1976;34:403).”
Further, very few cryoprotectants are available to store platelets at temperatures below 0° C. Known cryoprotectants are dimethylsulfoxide (DMSO), hydroxyethyl starch (HES), polyethylene glycol (PEG) and glycerol. These cryoprotectants are usually added to plasma containing platelets. Because they either interfere with the blood coagulation mechanism or are toxic, they are usually removed from the platelet suspension before the transfusion (Transfusion Medicine Reviews, 2003, 17: 263-271).
It has been reported that DMSO is the best of these three options as it best preserves platelet morphology and function (Rothwell et al. 2000 Transfusion, 40:988-993). A concentration of 5-6% (w/v) DMSO provides the best results for long-term storage of platelets at −80° C. However, since DMSO is very toxic, removal of the cryoprotective agent present in the thawed cell suspension is a necessary step before transfusion of cryopreserved platelets. This process, currently performed by centrifugation, is labor intensive and negatively affects platelet viability. Indeed, cells washed by centrifugation, which results in a pellet, must be left undisturbed to give the cells time to recover from the washing before resuspension. It has been shown that the release of PF4 and the expression of CD62P are significantly higher with centrifuged platelets, which are signs of platelet activation. In a nutshell, DMSO could be an appropriate cryoprotectant but since it has to be removed from the platelet suspension prior to transfusion, it increases the risk for bacterial contamination and adds a mechanical stress on the platelets.
With respect to the storage and freezing of platelets, it is known in the art that in vitro results are predictive of in vivo functionality (Rothwell et al. 2000 Transfusion 40: 988-993). It is also recognized in the art that it is not necessary for frozen and thawed platelet substitutes to retain 100 percent of in vitro function to have satisfactory results in vivo (Rothwell et al. 2000 Transfusion 40: 988-993).
Thus, there is a need for a method for storing platelets at temperatures below 4° C. such that the stored/thawed platelets have acceptable platelet functionality and viability. There is also a need for a non-toxic cryoprotectant.